The present invention relates to an apparatus and a method for determining the concentration of a substance. In one particular application, the invention relates to apparatus and a method for determining the concentration of oxygen within living tissue cells.
For clarity, the term xe2x80x9cassay substancexe2x80x9d will be used in this specification to refer to the particular substance that an apparatus or method according to the invention is intended to detect.
It is well-known that a large number of fluorescent dyes measurably change their fluorescing characteristics in dependance upon the concentration of specific substances. For example, it is known that the fluorescence of the fluorophor tris(4,7-diphenyl-1,10-phenanthroline) ruthenium chloride is decreased in an inverse relationship to concentration of oxygen. It is recognised that both the intensity and the duration of light emitted by fluorescence of this substance are reduced in the presence of oxygen.
This phenomenon has been applied in measurement of oxygen tension in tumours, for example, as disclosed in the paper of W. K. Young, B. Vojnovic and P. Wardman: xe2x80x9cMeasurement of oxygen tension in tumours by time-resolved fluorescencexe2x80x9d British Journal of Cancer (1996) 74 (Supl.XXVII) S256-S259. In this disclosure, apparatus for measuring oxygen tension comprises a sensor in which a fluorophor is localised in a polymer and coated on an end surface of an optical fibre. A pulsed laser is used to apply pumping light to the fibre, which light is transmitted to the fluorophor. Following the pulse, the fluorophor emits light, which travels back along the fibre to a detector. An assessment of the concentration of oxygen present is made on the basis of the time taken for the fluorescent emission to decay, the rate of decay increasing with the concentration. This sensor is advantageous in that it is relatively unreactive with biological tissue and will not affect living cells even after a prolonged contact with them, and in that it does not consume oxygen during detection.
The operation of such apparatus is, to a large extent, satisfactory. However, the apparatus does have a major disadvantage. In order to generate fluorescence of sufficient intensity to be measurable, a high-intensity light source is needed. In practice, it has been found that a laser source is required. While laser sources are readily available, they are relatively costly, such that they represent a significant portion of the total cost of a sensor.
It is an aim of the invention to provide a system for detecting the concentration of an assay substance which has all of the advantages of the above-described prior art system, but which does not require use of a laser light source.
According to a first of its aspects, the invention provides a sensor for detecting concentration of an assay substance comprising an optical fibre having an end surface on which is disposed a polymer body within which a multiplicity of particles is immobilised, on which particles is adsorbed a fluorophor, the polymer being such as to allow the assay substance to permeate into the body to come into contact with the fluorophor, and the fluorophor being selected as having a fluorescent activity which is measurably altered in the presence of the assay substance.
It has been found that the pattern of distribution of the fluorophor in a sensor embodying the invention is particularly effective in transmitting a high proportion of the light generated by the fluorophor back along the fibre for detection.
Preferably, the sensor comprises a single optical fibre which carries light from a pumping light source to the polymer body and which carries light emitted by the fluorophor from the polymer body to a detector. This further simplifies the construction of the sensor.
It has been found to be advantageous for the numeric aperture of the optical fibre to be greater than 0.3, and, in some embodiments, yet more advantageous to be greater than 0.4. In particularly preferred embodiments, a numerical aperture on the range 0.45 to 0.5 is selected. Numerical apertures in these ranges are particularly suited to collection of light from the fluorophor in the body of active material.
An optical fibre for use in a sensor embodying the invention is advantageously of less than 300 xcexcm in diameter. A diameter of approximately 200 xcexcm has been found to be particularly suitable.
The particles may suitably be silica gel particles. It is generally preferable for such particles to be of as small a size as possible, since this maximises the surface area on which dye can be adsorbed and also promotes cross-linking of the polymer matrix. At present, silica particles having an average diameter of 5 xcexcm are readily available. It is preferable that the silica particles are of an average diameter of 5 xcexcm or less.
In a particularly useful embodiment of the invention, the assay substance is oxygen. In such embodiments, a suitable polymer is a silicone rubber. A suitable fluorophor for use in such embodiments is a ruthenium complex dye, for example tris(4,7-diphenyl-1,10-phenanthroline). However, many other dyes could also be used, a prime desirable property being that the dye has a relatively long fluorescent lifetime; preferably in the order of several xcexcs when unquenched by the assay substance.
In principle, a dye with a short fluorescent lifetime could be used. If this is the case, a correspondingly fast light source must be used to excite the fluorophor, and the light source must be controlled by suitably fast switching circuitry.
From another of its aspects, the invention provides a method of making a sensor for measuring the concentration of an assay substance in which a fluorescent dye is adsorbed onto a multiplicity of solid particles, subsequently dispersing the particles in a liquid polymer, applying the liquid polymer and the particles contained in it to an end surface of an optical fibre, and curing the liquid polymer to form a polymer body on the end surface of the optical fibre, the dye having been selected as to have a fluorescent activity which is measurably altered in the presence of the assay substance, and the polymer having been selected to be permeable to the assay surface when in its cured condition.
In such methods, the optical fibre typically comprises a core, a cladding, and a protective buffer coating which covers the cladding. In such cases, the method includes removal of the buffer coating from an end portion of the fibre prior to application of the liquid polymer, and subsequent to curing of the liquid polymer, a protective coating is applied to cover the said end portion and the polymer body. In such embodiments, the protective coating may be formed from the same liquid polymer as is applied to the end portion of the fibre. The polymer of the protective coating may be substantially pure or it may incorporate opaque particles such as carbon black. This latter arrangement isolates the fluorophor from ambient light, while the former arrangement may offer greater acceptability for use in contact with biological tissue.
Alternatively, the method may include insertion of the said end portion through a hollow member, such that its end face, on which the liquid polymer is applied, projects from the hollow member, and subsequent to curing of the polymer layer, a protective coating is applied to cover an end face of the tubular member and the polymer body. The hollow member may be a hollow needle made, for example, from steel or a ceramic material. The arrangement described in this paragraph has the advantage of having high mechanical durability.
In a third of its aspects, the invention provides a system for measuring the concentration of an assay substance comprising:
a sensor having a sensing body including fluorophor, which fluorophor has a fluorescent activity which is measurably altered in relation the concentration of the assay substance and light conveying means for conveying light to and from the fluorophor;
a pumping light source which, in operation, applies light to the light conveying means to activate the fluorophor;
a detector for detecting light emitted by the fluorophor in the polymer body and for generating a signal in response thereto;
analysing apparatus for analysing the signal generated by the detector, and calculating from that signal the concentration of assay substance detected by the sensor; characterised in that
the detector operates to detect transient change in light emitted by the fluorophor simultaneously with the pumping light source operating to apply light to the optical fibre.
This system is particularly advantageous because operation of the light source simultaneously with the detector results in a substantially greater amount of fluorescent light output, than would occur with a pulsed source of an equivalent brightness operated briefly before operation of the detector. By virtue of this, the light source may be a source other than a laser, for example, one or more light emitting diodes.
A system according to this aspect of the invention may operate by analysing the change in the light emitted by the fluorophor which occurs after the light source starts to operate. As such, the system analyses the kinetics of the growth in light output which takes place in response to operation of the light source. In such a system, it is normal to operate the light source for a time which is substantially larger than the time during which light emitted by the fluorophor changes following the start of operation of the light source.
In most practical embodiments, the light source is operated repeatedly, the analysing means being operable to calculate an average value of a plurality of calculated concentration values. This arrangement ensures that random variations in any one measurement do not substantially affect the accuracy of the system.
It is preferable in a system according to this aspect of the invention for the sensor to have a single optical fibre through which light is transmitted to the fluorophor, and through which light from the fluorophor is returned to the detector. In such embodiments, the system typically further comprises an optical means, such as beam splitter, to split light emerging from the optical fibre from light which is entering the fibre from the light source.
In another of its aspects, the invention provides an analysis system for calculating a lifetime period of an exponentially varying signal comprising:
three or more integrating circuits, each having an input for receiving the signal an output on which is generated a signal representative of the value of the input signal integrated over time;
for each integrator, a switch circuit having a control input and operative to selectively connect or disconnect the input of the associated integrator to the signal in dependance upon the state of its control input;
timing means responsive to commencement of exponential variation of the signal, and operative to generate control signals for application to the control inputs of the switch circuits, the control signals being timed such that the three switches connect their respective integrating circuits, in turn, to the signal for three equal and consecutive time periods during exponential change of the signal; and
computing means operative to receive output signals from the integrating circuits, and perform on them analysis whereby the lifetime value of the exponential change can be determined.
This system is particularly useful in systems of the above defined aspects of the invention, but can also find application in other systems in which high-speed analysis of exponentially varying signals is needed. Its particular advantage is that the mathematically difficult task of performing the integration is carried out in high-speed, low-cost dedicated hardware. The computing means can therefore be of relatively low performance, since it need perform only a few mathematically simple operations.
The integrating circuits can conveniently be implemented as operational amplifier integrators.
It may be that alternative faster integrating circuits would be required if a dye of relatively short fluorescent lifetime is used.
The computing means will typically comprise a digital computer, with a suitable analogue-to-digital conversion circuit being provided between the integrating circuits and the computing means. In particularly convenient embodiments, the computing means comprises a general-purpose, microprocessor-based computer, such as a desktop personal computer.